title: An antibody targeted to VEGFR-2 Ig domains 4-7 inhibits VEGFR-2 activation and VEGFR-2-dependent angiogenesis without affecting ligand binding.
abstract: Inhibition of VEGFR-2 signaling reduces angiogenesis and retards tumor growth. Current biotherapeutics that inhibit VEGFR-2 signaling by either sequestering VEGF ligand or inhibiting VEGF binding to VEGFR-2 may be compromised by high VEGF concentrations. Here we describe a biotherapeutic that targets VEGFR-2 signaling by binding to Ig domains 4-7 of VEGFR-2 and therefore has the potential to work independently of ligand concentration. 33C3, a fully human VEGFR-2 antibody, was generated using XenoMouse technology. To elucidate the mechanism of action of 33C3, we have used a number of competition and binding assays. We show that 33C3 binds VEGFR-2 Ig domains 4-7, has no impact on VEGF-A binding to VEGFR-2, and does not compete with an antibody that interacts at the ligand binding site. 33C3 has a high affinity for VEGFR-2 (K(D) < 1 nmol/L) and inhibits VEGF-A induced phosphorylation of VEGFR-2 with an IC(50) of 99 ± 3 ng/mL. In vitro, in a 2D angiogenesis assay, 33C3 potently inhibits both tube length and number of branch points, and endothelial tubule formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model. These data show targeting VEGFR-2 outside of the ligand binding domain results in potent inhibition of VEGFR-2 signaling and inhibition of angiogenesis in vitro and in vivo.
authors.affiliation: Cancer Bioscience, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, United Kingdom.
authors.forename: Jane
authors.initials: J
authors.lastname: Kendrew
authors.forename: Cath
authors.initials: C
authors.lastname: Eberlein
authors.forename: Brad
authors.initials: B
authors.lastname: Hedberg
authors.forename: Karen
authors.initials: K
authors.lastname: McDaid
authors.forename: Neil R
authors.initials: NR
authors.lastname: Smith
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Weir
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Wedge
authors.forename: David C
authors.initials: DC
authors.lastname: Blakey
authors.forename: Ian
authors.initials: I
authors.lastname: Foltz
authors.forename: Joe
authors.initials: J
authors.lastname: Zhou
authors.forename: Jaspal S
authors.initials: JS
authors.lastname: Kang
authors.forename: Simon T
authors.initials: ST
authors.lastname: Barry
mesh_terms: D020533:Angiogenesis Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000378:metabolism* / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D016513:Mice, SCID; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012867:Skin / Q000098:blood supply* / Q000187:drug effects; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020533:Angiogenesis Inhibitors; D000906:Antibodies; D008024:Ligands; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2